CN-122016946-A - Non-targeted single-molecule conductivity spectrum measuring device, device and method

CN122016946ACN 122016946 ACN122016946 ACN 122016946ACN-122016946-A

Abstract

The invention provides a non-targeted single-molecule conductivity spectrum measuring device, a device and a method, which are based on a tunneling device of integrated dielectrophoresis forceps, and the tunneling device is used for performing dielectrophoresis capturing and enrichment with extremely low concentration on molecules and tunneling electrical signal detection, namely homobody capturing and detection in a complex matrix represented by urine. The detection target does not need to be predefined before detection. The collection of various molecular information in the urine matrix is realized through the strong capture and enrichment capability of dielectrophoresis forceps. The device can be used for obtaining tunneling electrical signal spectrums corresponding to different urine samples, then carrying out multidimensional feature extraction on the signal spectrums, and realizing accurate classification of different samples through a machine learning model.

Inventors

TANG LONGHUA
WANG ZIAN
YANG YUXIN
YI LONG
LIU XU

Assignees

浙江大学

Dates

Publication Date: 20260512
Application Date: 20260416

Claims (10)

1. The non-targeted single-molecule conductivity spectrum measuring device is characterized by comprising a single-molecule enrichment unit and a tunneling detection unit, wherein the single-molecule enrichment unit is used for capturing, enriching or positioning single molecules in a sample to be measured to a nano gap or near the nano gap, the tunneling detection unit is used for performing non-targeted detection on the single molecules entering the nano gap, and the single-molecule enrichment unit has nano-scale control precision and comprises any one or more of dielectrophoresis, electrophoresis and electrochemical tweezers.
2. The measurement device of claim 1, wherein the single molecule enrichment unit is a dielectrophoresis manipulation unit and comprises 1-4 auxiliary electrodes, the tunneling detection unit comprises a pair of nanogap electrode pairs, the gap width of the nanogap electrode pairs is sub-5 nanometers, the surfaces of the nanogap electrode pairs are modified with anti-adsorption molecule layers of cysteine or mercaptoethanol, and the auxiliary electrodes and the nanogap electrode pairs are arranged orthogonally, in a nested or adjacent mode in space.
3. The measuring device of claim 2, further comprising an insulating substrate, wherein the dielectrophoresis manipulation unit and the tunneling detection unit are insulated from each other by the insulating substrate, and wherein the insulating substrate is made of any one or more of quartz, a glass tube, a silicon wafer, a glass sheet, a high molecular polymer, and a flexible material.
4. A measurement device for non-targeted single molecule conductivity spectrum, characterized by comprising the measurement device, the microfluidic channel sample cell and support and the electrical control module according to any one of claims 1-3.
5. The measuring device of claim 4, wherein the microfluidic channel sample cell comprises a glass tube with an inner diameter of 1.5 mm to 2.5 mm and a support frame thereof, wherein the support frame is used for obliquely placing the glass tube at an angle of 10-30 degrees, and the microfluidic channel sample cell further comprises an electrical control module comprising a working electrode, a reference electrode, a function signal generator, a patch clamp amplifier and a digital-to-analog converter.
6. Use of a measuring device according to any one of claims 1 to 3 or a measuring apparatus according to any one of claims 4 to 5 for non-targeted detection of complex substrates.
7. A method for measuring a non-targeted single-molecule conductivity spectrum, which is characterized in that the measuring device as claimed in any one of claims 1 to 3 or the measuring device as claimed in any one of claims 4 to 5 is adopted for detection, a sample to be measured is contacted with the measuring device in a microfluidic channel sample cell, the conductivity spectrum characterization is obtained, and the non-targeted characterization of single molecules in the sample to be measured in situ and in real time electronics is realized.
8. The measurement method according to claim 7, characterized in that the method comprises the steps of: (1) The method comprises the steps of (1) enabling a sample to be detected and a measuring device to contact in a microfluidic channel sample cell, enabling tunneling detection units to be in an electrified state, starting a dielectrophoresis operation unit to apply an alternating current electric field, and driving and enriching molecules in the sample into a detection gap; (2) Closing a dielectrophoresis control unit, acquiring a non-targeted single-molecule conductivity spectrum of a sample to be detected through a tunneling detection unit, and alternately executing the step (1) and the step (2) for more than 4 times so as to maintain the molecule capturing probability in a detection gap and accumulate single-molecule conductivity spectrum data; (3) And (3) through analysis and feature extraction, constructing a model to realize classification of the sample to be detected.
9. The measurement method of claim 8, wherein the sine alternating current signal peak value of the dielectrophoresis operation unit is 1-20 Vpp, the frequency is 100 khz-5 MHz, the time for applying the alternating current electric field in the step (1) is 5-20 s, the bias voltage applied to the tunneling detection unit is 50-300 mV in the step (2), and the collection time of the non-targeted single-molecule conductivity spectrum of the sample to be measured is 4-10 min.
10. The method of claim 9, wherein the features include feature parameters extracted in multiple dimensions including time domain statistics, fourier spectrum features, power spectrum features, wavelet features, and nonlinear features, the sample to be measured is any one or more of urine, serum, saliva, milk, or wine, and the classification of the sample to be measured includes any one or more of disease-to-health differentiation, differentiation of different disease types, pathogen identification, and cancer typing.

Description

Non-targeted single-molecule conductivity spectrum measuring device, device and method Technical Field The invention relates to the technical field of single-molecule detection and micro-nano device intersection, in particular to a non-targeting single-molecule conductivity spectrum measuring device, a non-targeting single-molecule conductivity spectrum measuring device and a non-targeting single-molecule conductivity spectrum measuring method. Background Early diagnosis of disease is critical to prognosis. At present, detection of molecular markers in complex matrixes such as urine and the like has proved to be an effective path for early diagnosis of diseases, and is a key requirement in the fields of modern clinical diagnosis, life science research, environmental monitoring and the like, however, the matrix components are extremely complex, contain high-concentration electrolytes, proteins, metabolites and cell fragments, and pose serious challenges to the sensitivity, specificity and anti-interference capability of detection technologies. At present, in the conventional detection and analysis method, physicochemical and morphological analysis is used as a clinical basic detection means, an automatic biochemical analyzer is used for detecting the pH value, osmotic pressure and electrolyte concentration of a sample, and meanwhile, the flow cytometry or microscopic examination is used for counting and morphological observation of formed components such as cells, tubes and crystals in the sample, so that macroscopic index reference is provided for preliminary diagnosis; the immunological detection technology is based on the specific recognition principle of antigen-antibody, and covers classical enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), colloidal gold lateral chromatography, electrochemiluminescence (ECL) and other technologies, and is widely applied to quantitative detection of specific protein, hormone or drug molecules in complex matrixes, the chromatography and mass spectrometry technology is used as an advanced means for analysis of complex matrix components, separation is realized by Gas Chromatography (GC) or Liquid Chromatography (LC) according to the physicochemical properties of the components, such as polarity, molecular weight and the like, and then the precise identification is carried out by combining a Mass Spectrometer (MS) according to mass-to-charge ratio, thus being a core method for metabonomics and proteomics research, the nucleic acid amplification and analysis technology is used for carrying out sequence analysis and quantification on trace nucleic acids (such as cfDNA, miRNA and the like) in samples by adopting Polymerase Chain Reaction (PCR), isothermal amplification technology or second-generation sequencing (NGS), and the spectrum analysis technology is used for providing support for diagnosis of gene-related diseases, the spectrum analysis technology is used for utilizing the selective absorption, scattering or emission characteristics of electromagnetic waves by using the molecular spectrum, ultraviolet-visible absorption spectrum (UV-Vis), infrared spectrum (IR), fluorescence spectrum and other technologies, the overall chemical composition or specific functional group distribution of the matrix is evaluated. The Single-Molecule conductivity spectrum detection is an ultra-high sensitivity characterization technology for researching electron transport characteristics on a Single Molecule level, has the remarkable advantages of no need of marking, high response speed, high signal to noise ratio and capability of directly revealing the molecular mass energy level structure through electron transport fingerprints, and is characterized in that a nano-scale metal electrode gap is constructed, an electrical signal when a Molecule passes through or bridges the gap is recorded, and representative technical paths comprise a scanning tunneling microscope (STM-BJ) technology, a mechanically controllable cleavage technology (MCBJ) and a Single-Molecule electric field effect transistor (Single-molecular FET). The method comprises the steps of repeatedly carrying out 'contact-stretching' circulation on a substrate by means of a probe of a scanning tunnel microscope, capturing a conductive signal of a Molecule to be detected at the moment that an electrode is about to break, obtaining a conductive fingerprint spectrum of the Molecule through statistical analysis, carrying out micro-nano mechanical displacement control on a metal nanowire on a flexible substrate by a mechanical controllable splitting technology (MCBJ), breaking the metal nanowire through bending stretching to generate an atomic-level gap, and connecting a Single Molecule serving as a semiconductor channel between a source electrode and a drain electrode by a Single-Molecule electric field effect transistor (Single-Molecule FET), wherein the mechanical stability is extremely high, the Singl